Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The organisms which cause the disease known as "coccidiosis" in chickens belong to the phylum Apicomplexa, class Sporozoa, subclass Coccidia, order Eucoccidia, suborder Eimeriorina, family Eimeriidae, genus Eimeria. Within the Eimerian genus there are many species, several of which are pathogenic in chickens. The species of major concern to the chicken industry are Eimeria tenella, Eimeria maxima, Eimeria acervulina, Eimeria necatrix and Eimeria brunetti.
Coccidiosis has become a major economic problem in the chicken industry over the past few decades, mainly due to the overcrowding of chicken houses and drug resistance. The rearing of chickens under crowded conditions on a litter floor provides optimal conditions for the growth and spread of coccidia. Under such circumstances, sanitary control becomes nearly impossible and the farmer must rely on the effectiveness of coccidiostat drugs. However, drugs must be kept in the feed at all times and therefore are very expensive, certain drugs have costly side effects, and drug resistance has become a major problem under field conditions. Several large suppliers of these agents have come to realize that perhaps the only viable approach to the control of coccidiosis is by vaccine development.
The Eimerian parasites undergo a complex life cycle in the mucosa of the intestinal tract. They show a great deal of specificity both in terms of the species they infect and in their location within the intestine. In fact, site specificity of infection is used as the major criterion for diagnosis. Other parameters for diagnosis include size and shape of oocysts, characteristics of the infected intestine, weight loss, and skin pigmentation changes.
The life cycle of Eimeria is very similar to that of the hemosporidian parasites (i.e. plasmodium) except for the lack of an arthropod vector. Oocysts sporulate on the litter floor producing four sporocysts, each containing two sporozoites. These are ingested by the chicken and the sporozoites are released by the mechanical grinding of the oocysts in the gizzard and the subsequent digestion of the sporocyst wall by proteolytic enzymes. Sporozoites then invade lymphocytes and go on to invade epithelial cells where the asexual cycle begins. The parasite goes through 2-4 cycles of replication and division (each species having a defined number of divisions) leading to a production of large numbers of merozoites. After the final cycle of merozoite production the sexual cycle begins with the production of macrogametocytes (female) and microgametocytes. The macrogametocyte is characterized by the production of wall forming bodies which are involved in the production of the oocyst wall. Microgametocytes contain the components involved in the formation of microgametes which bud off from the surface of the intracellular parasite. Microgametes are flagellated and are responsible for the fertilization of the macrogamete. A zygote is formed which matures into the oocyst by fusion of the wall forming bodies and condensation of the nucleus. Oocysts are secreted in the feces, thus completing the cycle.
A single infection with Eimeria can lead to protection against subsequent reinfection with the same strain of that species. This finding was used as the basis for producing a live vaccine against coccidiosis. Small numbers of oocysts from all the major pathogenic species are provided either in water (COCCIVAC.TM.) or are encapsulated in a water soluble polysaccharide (British patent GB 2,008,404A) giving rise to subclinical infections. However, even with small numbers of oocysts severe outbreaks of coccidiosis have occurred and farmers are reluctant to introduce live, pathogenic parasites into their chicken houses.
In order to solve the problem of introducing pathogenic coccidia into a live vaccine, several laboratories have been working towards the production of live attenuated vaccines. By repeated passages in egg embryos or isolation of precocious lines (i.e. parasites that go through the life cycle in five days rather than six or seven), much less virulent and nonpathogenic strains of most of the major species have been isolated. Good protection has been observed using this approach, however there are still problems which include: shelf life of live parasites; back mutations of attenuated lines to pathogenic strains; strain variation especially in the case of Eimeria maxima; inability to protect for long periods of time; and introduction of any live coccidia into a chicken house would be resisted by farmers. With all these problems, the attenuated live vaccine may be used in the field until a more defined, noninfective, subunit vaccine can be developed.
In the area of subunit vaccines the extracellular stages of the life cycle (the sporozoite, the merozoite and the gametes--micro and macro) are the most vulnerable to immune attack. The sporozoite is the first stage of development after the parasite is released from the oocyst and after a short time in the lumen invades a lymphocyte. In natural infections, high titers of antibody to sporozoites have been found and this stage is considered to be most promising for vaccine development. As a result, several laboratories have been working towards a sporozoite vaccine.
E. tenella sporozoite extracts as well as supernatants of ground, sporulated oocysts were shown to protect broilers up to 7 weeks of age from challenge infections (1). In work using monoclonal antibodies, results have indicated that preincubation of sporozoites with monoclonal antibodies directed against surface antigens and injection into the cloaca of chickens, can give partial protection against the infection. Antigens recognized by these monoclonal antibodies have been identified by Western blotting and one of them, a protein of 25,000 molecular weight, has been cloned and sequenced (European patent publication No. 0 164 176, published Dec. 11, 1985). This antigen was tested as an immunogen for protection against challenge infections where partial immunity was observed. American Cyanamid has also made several monoclonal antibodies to sporozoite surface antigens, some of which exhibited partial protection against Eimeria tenella infections (European patent publication No. 0 135 712, published Apr. 3, 1985, and European patent publication No. 0 135 073, published Mar. 27, 1985). The reason that purified antigens gave only partial protection in those studies may be due to the finding, that Eimeria tenella sporozoites can cap and shed their surface antigens, which is an important mechanism of host immune invasion (2). Thus, it appears that the sporozoite stage may contain some antigens which can give partial protection, however, it seems unlikely that full protection will be achieved using only a sporozoite vaccine.
The role of antibody in the protective immune response against Eimeria in chickens has received a great deal of attention. In several studies carried out by Rose & Long (3-8), it was found that serum taken from birds which had recovered from infections with E. tenella or E. maxima can give passive protection against challenge infections with these species. Based on these results and similar to the studies described above, Rose & Long immunized chickens with extracts of sporozoites and merozoites and the sera were tested for their effect on invasion by sporozoites in vitro and protection in vivo. In contrast to the active immunization results described above and in spite of the fact that these sera were capable of neutralizing sporozoites and merozoites in vitro, birds which were immunized with extracts of sporozoites or merozoites showed no resistance to oral infections with E. tenella oocysts. Furthermore, sera from the same immunized birds could not be used to protect naive chickens. Thus, those authors concluded that antibody may play only a minor role in protection against Eimeria in spite of their passive immunization results (3-8).
In previous reports maternal antibody was shown to play an important role in resistance to coccidial infection (4). In one experiment laying hens were given multiple infections with E. tenella and the offspring were challenged with oocysts from the same strain and compared with chicks from uninfected layers used as a control. It was found that the chicks hatched from immune layers had a 75% lower oocyst output as compared to the control. These results corroborate the passive immunization studies described by Rose and her coworkers (3-8), and based on the parameter of oocyst output show that maternal antibody plays an important role in protective immunity.
A vaccine using antigens from the merozoite stage is also being tested (European patent publication No. 0 135 073). Several laboratories are making monoclonal antibodies to merozoite surface antigens in order to test their ability to inhibit invasion in vitro and in vivo. Most of these antigens were also found to be present in sporozoites as well as in different Eimerian species (9,10). In vivo protection results using these monoclonal antibodies again only showed partial inhibition and only with very low numbers of parasites (challenge dosage of 200 sporozoites) (European patent publication No. 0,135,172). Similar studies have been carried out for malarial merozoite surface antigens where excellent results using monoclonal antibodies to inhibit invasion in vitro have been obtained (11,12), however poor results were found in vivo. Problems of antigenic variation and diversity at this developmental stage of malaria is probably the major reason. Currently, attempts are being made to identify constant epitopes within these antigens and produce small peptedes which contain the important antigenic determinants.
The gametocyte stages of Eimerian development are the most difficult to analyze and very little has appeared in the literature regarding gamete subunit vaccines due to the following reasons: despite much effort Eimerian gametes have never been isolated before and, therefore, new methods need to be developed in order to study this developmental stage at the molecular level; no efficient in vitro system has been described for working with the sexual stages of Eimerian development; and most previous reports by leading authors in the field have led to the conclusion that gametes play little or no role in protective immunity (13,14,30).
In work carried out by Rose on immunity to E. maxima (5-8), it was found that sera taken from recovered birds 14 days post infection can give passive protection of up to 93% (based on oocyst output) against challenge with E. maxima, and these sera by Ouchterlony were found to precipitate an antigen prepared from mucosa containing gametocyte stages and not with sporozoite proteins (8). However, in these studies no direct proof of gametes playing a role in protective immunity was reported, nor were experiments done using the recovered antisera to identify the antigens seen by Ouchterlony. In fact, gametocytes were not purified and tested; only a mucosal extract was tested. In subsequent work Rose and others did not conclude that sexual stages, i.e., gametes play any role in induction of protective immunity to E. maxima (14) or to any other Eimeria species in spite of the results they obtained. In order to assess the role of the recovered sera in protective immunity, studies at the molecular level are required to characterize the proteins being recognized and the particular stage(s) at which they are being synthesized.
In studies carried out on malarial gametocytes and gametes, it was found that antigens from these stages can be used to protect birds against transmission of the disease (15). Furthermore, monoclonal antibodies to these antigens can be used to block the further development of the malaria parasite (16) .
One of the prerequisites for studying the role of gametocytes in protective immunity, is their isolation. No prior report has been made on isolating gametocytes of Eimeria. Most of the published work done on gametocytes prior to the subject invention involved using electron microscopy to observe the growth and development of gametocytes in vivo. No studies have been published on the identification of stage specific gametocyte antigens. In early attempts to analyze gametocyte antigens in either whole infected intestine or partially purified preparations, little or no difference was seen by SDS polyacrylamide gel electrophoresis between infected intestine and normal intestine. It was therefore necessary for us to develop a method for isolation of gametocytes to a very high degree of purity in order to carry out molecular studies.
Once gametocytes are isolated they can be used to identify and isolate the protective transmission blocking antigens of Eimeria maxima. Monoclonal and polyclonal antibodies can be raised against these antigens and used to passively protect chicks as well as to identify cDNA clones in a gametocyte cDNA expression library. Once the antigens are isolated (either native or cloned) they can be tested for their ability to elicit protective immunity in young chicks.
One of the difficulties in vaccinating young chicks however, is the lack of a mature immune system capable of responding to the administered antigens in a subunit vaccine. Another approach whereby large amounts of specific antibody can be provided to newborn chicks is through maternal immunization of laying hens. Gametocyte antigens can be used to immunize laying hens who thereby provide large amounts of specific protective anti-gametocyte antibody to their offspring chicks via yolk antibodies. Furthermore, by providing such antibody against gametocyte antigens, newborn chicks are susceptible to infection by the asexual stages of development. These asexual stage antigens act to induce protective immunity against invasion by sporozoites and merozoites in subsequent reinfection, while the anti-gametocyte antibodies act to reduce oocyst output by blocking either the growth, development and/or fertilization of gametocytes.
The concept of using defined coccidial antigens to immunize chicks against infection via maternal immunity is a novel one. The work described above by Rose & Long on maternal immunity (4), only relates to the effect of live infections on laying hens and transfer of resistance via maternal immunity to the chicks. In those studies no mention was made of the use of native or cloned stage specific antigens to induce protective immunity, nor was the mechanism by which the live infection induced protection in offspring chicks analyzed.
The present invention involves a method for the isolation of E. maxima macrogametocytes and microgametocytes. The isolation procedure of the present invention allows for the purification, to a very high degree (over 90%) of E. maxima gametocytes, with little or no host contamination (based on SDS-polyacrylamide gel electrophoresis analysis).
The present invention also concerns the identification of protective gametocyte antigens and the RNA which encodes them. These antigens were found to give partial protection against challenge infections with E. maxima in vivo. Antisera from immunized and recovered chickens as well as immunized mice, guinea pigs, and rabbits, have been used to identify the antigens involved by Western blotting and immune precipitation techniques. These antisera as well as monoclonal antibodies and soybean lectin were used to isolate the antigens from protein extracts of gametocytes, as well as to screen a cDNA expression library prepared from gametocyte mRNA. In addition, these antibodies were used to passively immunize chickens against challenge infections, while the antigens were used to actively immunize chicks.
The present invention provides a method of conferring upon a newborn chick maternal immunity (antibodies) against infection and/or transmission of an Eimeria spp. which comprises administering to a laying hen at a suitable time prior to the hen laying a fertilized egg an amount of a native or recombinant antigenic protein present in gametocytes of the Eimeria spp. effective to induce in the hen an immune response conferring protection via maternal immunity against infection and/or transmission of an Eimeria spp. in the offspring chick.